The inhibitor of DNA binding protein ID3 has been associated with the progression of colorectal cancer (CRC). Despite its significance, its specific role in the immune evasion strategies utilized by CRC remains unclear. RNA-seq analysis revealed that ID3 was positively associated with the PD-L1 immune checkpoint. We further demonstrated that tumor cell-expressed ID3 enhanced PD-L1 expression, suppressed the infiltration and activation of CD8+ T cells, and facilitated the immune evasion of CRC cells. Additionally, we found that knockdown of ID3 significantly enhanced the effectiveness of PD-L1 antibody blockade treatment in combating CRC, reduced the upregulation of PD-L1 induced by the antibody, and altered the immune microenvironment within CRC. Mechanistically, ID3 interacted with the transcription factor MYC and reconstructed the four-dimensional structure of MYC, thereby enhancing its binding affinity to the PD-L1 promoter and augmenting PD-L1 transcriptional activity. By integrating analysis of ChIP-seq, RNA-seq, and ImmPort gene sets, we found that ID3's DNA-assisted binding function was widespread and could either enhance or suppress gene transcription, not only affecting tumor immune escape through immune checkpoints but also regulating various cytokines and immune cells involved in tumor immunity. In conclusion, our study uncovers a mechanism by which ID3 promotes immune evasion in CRC and implicates that targeting ID3 may improve the efficacy of anti-PD-1/PD-L1 immunotherapy.

IntroductionHepatocellular carcinoma (HCC) represents a significant health challenge, with immunotherapy serving as a crucial component of its complex treatment regimen. This study investigates the use of TP53Y220C as a preferred antigen to induce cytotoxic T lymphocytes (CTLs) for cytotoxic effects against HCC.MethodsThe TP53Y220C mRNA (mTP53Y220C) was synthesized through an in vitro transcription method and subsequently introduced into dendritic cells (DCs) using bacterial outer membrane vesicles expressing L7Ae and Listeria monocytogenes lysin O (OMV-LL), electroporation, and lipid nanoparticles, respectively. Co-culture of differently treated DCs with initial T cells induces CTLs. The cytotoxic effects of CTLs on hepatocellular carcinoma were evaluated through experiments such as flow cytometry and mouse tumour models.ResultsWe assessed the therapeutic efficacy of CTLs, activated by mTP53Y220C-loaded DCs, in a murine model of HCC. Results demonstrate that CTLs, activated by DCs loaded with mTP53Y220C via OMV-LL or electroporation, effectively initiated immune responses against HCC. While OMV-LL were less efficient than electroporation in mRNA delivery, they induced a significant pro-inflammatory response and activated the innate immune system.ConclusionThis study highlights OMV-LL as an innovative mRNA delivery approach to DCs for CTLs activation and demonstrates their potential in CTLs-based therapy for HCC.

BACKGROUND:Cellular senescence (CS) is thought to be the primary cause of cancer development and progression. This study aimed to investigate the prognostic role and molecular subtypes of CS-associated genes in gastric cancer (GC). MATERIALS AND METHODS:The CellAge database was utilized to acquire CS-related genes. Expression data and clinical information of GC patients were obtained from The Cancer Genome Atlas (TCGA) database. Patients were then grouped into distinct subtypes using the "Consesus- ClusterPlus" R package based on CS-related genes. An in-depth analysis was conducted to assess the gene expression, molecular function, prognosis, gene mutation, immune infiltration, and drug resistance of each subtype. In addition, a CS-associated risk model was developed based on Cox regression analysis. The nomogram, constructed on the basis of the risk score and clinical factors, was formulated to improve the clinical application of GC patients. Finally, several candidate drugs were screened based on the Cancer Therapeutics Response Portal (CTRP) and PRISM Repurposing dataset. RESULTS:According to the cluster result, patients were categorized into two molecular subtypes (C1 and C2). The two subtypes revealed distinct expression levels, overall survival (OS) and clinical presentations, mutation profiles, tumor microenvironment (TME), and drug resistance. A risk model was developed by selecting eight genes from the differential expression genes (DEGs) between two molecular subtypes. Patients with GC were categorized into two risk groups, with the high-risk group exhibiting a poor prognosis, a higher TME level, and increased expression of immune checkpoints. Function enrichment results suggested that genes were enriched in DNA repaired pathway in the low-risk group. Moreover, the Tumor Immune Dysfunction and Exclusion (TIDE) analysis indicated that immunotherapy is likely to be more beneficial for patients in the low-risk group. Drug analysis results revealed that several drugs, including ML210, ML162, dasatinib, idronoxil, and temsirolimus, may contribute to the treatment of GC patients in the high-risk group. Moreover, the risk model genes presented a distinct expression in single-cell levels in the GSE150290 dataset. CONCLUSION:The two molecular subtypes, with their own individual OS rate, expression patterns, and immune infiltration, lay the foundation for further exploration into the GC molecular mechanism. The eight gene signatures could effectively predict the GC prognosis and can serve as reliable markers for GC patients.

High-frequency mutation of the TP53 tumor suppressor gene is observed in multiple human cancers, which promotes cancer progression. However, the mutated gene-encoded protein may serve as a tumor antigen to elicit tumor-specific immune responses. In this study, we detected widespread expression of shared TP53-Y220C neoantigen in hepatocellular carcinoma with low affinity and low stability of binding to HLA-A0201 molecules. We substituted the amino acid sequences VVPCEPPEV with VLPCEPPEV in the TP53-Y220C neoantigen to yield a TP53-Y220C (L2) neoantigen. This altered neoantigen was found to increase affinity and stability and induce more cytotoxic T lymphocytes (CTLs), indicating improvements in immunogenicity. In vitro assays showed the cytotoxicity of CTLs stimulated by both TP53-Y220C and TP53-Y220C (L2) neoantigens against multiple HLA-A0201-positive cancer cells expressing TP53-Y220C neoantigens; however, the TP53-Y220C (L2) neoantigen showed higher cytotoxicity than the TP53-Y220C neoantigen against cancer cells. More importantly, in vivo assays demonstrated greater inhibition of hepatocellular carcinoma cell proliferation by TP53-Y220C (L2) neoantigen-specific CTLs relative to TP53-Y220C neoantigen in zebrafish and nonobese diabetic/severe combined immune deficiency mouse models. The results of this study demonstrate enhanced immunogenicity of the shared TP53-Y220C (L2) neoantigen, which has the potential as dendritic cells or peptide vaccines for multiple cancers.

OBJECTIVE:We herein demonstrate the efficacy of PICC placement through a superficial femoral vein in patients with superior vena cava syndrome using ultrasound guidance and electrocardiographic localization. The treatment of PICC disconnection was also discussed.METHODS:The study enrolled 51 patients with superior vena cava syndrome. Ultrasound-guided technology and ECG positioning technology are employed to help these patients in catheterization. The puncture time, the number of punctures, and catheter tip position were recorded. The patient was followed up for at least 2 years. The complications and treatment during follow-up were recorded.RESULT:The average puncture time was 32.13 ± 3.91 min. A total of 49 patients were successfully punctured once, while 2 patients failed in the first puncture. The main reason for puncture failure is that the inability of a guide wire to pass through. After the nurse removed the needle and pressed the puncture point until no rebleeding occurred, the puncture above the original puncture point was successful. X-ray examination revealed that the catheter tip was located in the inferior vena cava, above the diaphragm, near the right atrium. The success rate of catheterization was 100%. The visual analog scale (VAS) score was (2.44 ± 0.73) at the time of puncture, which was tolerable during the operation, and the patient did not complain of obvious pain following the operation. One patient developed complications of broken tube half a year after the puncture. Interventional physicians utilized angiography to locate the broken catheter.CONCLUSION:It is safe and feasible to place PICC through a superficial femoral vein under ultrasound combined with ECG positioning technology in patients with superior vena cava syndrome.

The efficacy of conventional treatments for pancreatic cancer remains unsatisfactory, and immunotherapy is an emerging option for adjuvant treatment of this highly deadly disorder. The tumor-associated antigen (TAA) MUC1 is expressed in a variety of human cancers and is overexpressed in more than 90% of pancreatic cancer, which makes it an attractive target for cancer immunotherapy. As a self-protein, MUC1 shows a low immunogenicity because of immune tolerance, and the most effective approach to breaking immune tolerance is alteration of the antigen structure. In this study, the altered MUC1(1068-1076Y1) epitope (YLQRDISEM) by modification of amino acid residues in sequences presented a higher immunogenicity and elicited more CTLs relative to the wild-type (WT) MUC1(1068-1076) epitope (ELQRDISEM). In addition, the altered MUC1(1068-1076Y1) epitope was found to cross-recognize pancreatic cancer cells expressing WT MUC1 peptides in an HLA-A0201-restricted manner and trigger stronger immune responses against pancreatic cancer via the perforin/granzyme apoptosis pathway. As a potential HLA-A0201-restricted CTL epitope, the altered MUC1(1068-1076Y1) epitope is considered as a promising target for immunotherapy of pancreatic cancer. Alteration of epitope residues may be feasible to solve the problem of the low immunogenicity of TAA and break immune tolerance to induce immune responses against human cancers.

Chimeric antigen receptor (CAR) T cells have been proven effective for the treatment of B-cell-mediated malignancies. Currently, the development of efficient tools that supply CAR T cells for the treatment of other malignancies would have great impact. In this study, interleukin (IL)-15 and C-C motif chemokine ligand 19 (CCL19) were introduced into natural killer group 2D (NKG2D)-based CARs to generate 15×19 CAR T cells, which remarkably increased T-cell expansion and promoted the production of central memory T (Tcm) cells. 15×19 CAR T cells showed greater cytotoxicity to gastric cell lines than conventional CAR T cells and produced higher levels of IL-15 and CCL-19, which resulted in increased responder T cell chemotaxis and reduced expression of T cell exhaustion markers. A live zebrafish model was used for single-cell visualization of local cytotoxicity and metastatic cancers. Administration of 15×19 CAR T cells resulted in significant shrinking of gastric cancer xenograft tumors and expansion of 15×19 CAR T cells in zebrafish models. Taken together, these findings demonstrate that 15×19 CAR T cells are highly efficient in killing gastric cancer cells, are effective to avoid off-target effects, and migrate to local and metastatic sites for long-term surveillance of cancers.

BACKGROUND:Upregulated Kindlin-2 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. In this study, we investigated the molecular mechanism of Kindlin-2 in HCC.METHODS:Kindlin-2 downstream pathways were explored through microRNA sequencing. The Kindlin-2-miR-1258-TCF4 axis was verified using bisulfite sequencing, a luciferase reporter assay, quantitative real-time PCR, and rescue assays. Binding of TCF4 to the Kindlin-2 promoter was confirmed by promoter activity analysis and chromatin immunoprecipitation.RESULTS:MiRNA sequencing identified miR-1258 as a downstream effector of Kindlin-2. MiR-1258 expression was increased following Kindlin-2 knockdown and decreased after Kindlin-2 overexpression. Next, we identified transcription factor 7 like 2 (TCF7L2 or TCF4) as a target of miR-1258 and found that Kindlin-2 upregulated TCF4 expression by epigenetically suppressing miR-1258 in HCC. Furthermore, our results suggest that TCF4 binds to the Kindlin-2 promotor to enhance its transcription. Therefore, Kindlin-2-miR-1258-TCF4 interaction creates a positive feedback loop. Functional assays and animal experiments demonstrated critical roles of miR-1258 and TCF4 in HCC cell migration in vitro and HCC metastasis in vivo. In HCC tissues, Kindlin-2 expression correlated negatively with miR-1258 expression and positively with TCF4 expression. Meanwhile, miR-1258 expression correlated negatively with TCF4 expression.CONCLUSIONS:This study illustrates a novel integrin-independent signaling pathway, Kindlin-2-miR-1258-TCF4, that regulates HCC invasion and metastasis and identifies Kindlin-2 as a promising therapeutic target in HCC.

Background: Liver metastasis of colorectal cancer (CRC) remains high mortality and the mechanism is still unknown. Here we investigated the effects of inhibitor of DNA binding 2 (Id2) on growth and liver metastasis of CRC. Methods: qPCR and western blotting were used to demonstrate mRNA and protein expressions in Id2-knockdown HCT116 cells. Cell growth was observed by cell proliferation assay, colony formation assay and flow cytometry. Cell migration and invasion were observed with wound healing assay and transwell migration and invasion assay. The effects of Id2 knockdown on tumor growth and liver metastasis in vivo were evaluated respectively with subcutaneous tumor model and colorectal liver metastasis model by injecting HCT116 cells into the mesentery triangle of cecum in mice. Results: Id2 overexpression was found in CRC cell lines. Id2 knockdown resulted in a reduction in the proliferation, colony formation, migration and invasion of HCT116 cells. The suppression of cell proliferation was accompanied by the cell cycle arrest in the G0/G1 phase with down-regulation of Cyclin D1, Cyclin E, p-Cdk2/3, Cdk6, p-p27 and up-regulation of p21 and p27. Id2 knockdown reversed epithelial-mesenchymal transition (EMT) through increasing E-Cadherin and inhibiting N-Cadherin, Vimentin, beta-catenin, Snail and Slug. Id2 was also found to inhibit CRC metastasis via MMP2, MMP9 and TIMP-1. Furthermore, Id2 knockdown suppressed CRC liver metastasis in vivo. Conclusion: Id2 promotes CRC growth through activation of the PI3K/AKT signaling pathway, and triggers EMT to enhance CRC migration and invasion.

A method for the treatment of panniculitis caused by progesterone injection is introduced. Sixteen patients achieved good results. This is a 9-year single center retrospective study. Of all the 5633 patients who received progesterone injection, 16 developed panniculitis at the injection site. Pathological examination confirmed the occurrence of panniculitis. The patient received physical therapy. These treatments are determined by the course of the patient. Compared with patients without panniculitis, patients with panniculitis received more than one injection of progesterone. In 16 patients, symptoms and local signs disappeared completely in 15 patients. One patient did not take physical therapy according to the doctor's advice after the treatment improved. However, 1 month later, the patient went to see the doctor again and received the relevant physical therapy, and still achieved good results. Progesterone injection may lead to panniculitis, which is rare but may cause serious consequences. Physical therapy can be effective.

Colorectal cancer is the third most common cancer and the fourth leading cause of cancer-related mortality worldwide. Activating transcription factor 3 ( ATF3) has been identified as both an oncogene and a tumor suppressor in colorectal cancer, which encourages further studies to validate the exact role of ATF3 in colorectal cancer development and progression. The aim of this study was to examine the effect of ATF3 overexpression on biological behaviors of human colorectal cancer HCT116 cells. A stable ATF3-overexpressed HCT116 cell line was established by transfecting HCT116 cells with lentivirus carrying a full-length of ATF3. Cell proliferation was measured with MTS and colony-formation assays, and cell migration and invasion was detected using Transwell migration/ invasion assays. In addition, a sphere-forming assay was performed to assess the cancer-initiation capacity, while the gene and protein expression was quantified with qRT-PCR, Western blotting and flow cytometry. ATF3 overexpression was found to suppress proliferation, migration and invasion of HCT116 cells as compared with the control cells, while down-regulation of Survivin and PCNA expression was detected in HCT116 cells at both translational and transcriptional levels, and a reduction was measured in the CyclinD1 protein expression in HCT116 cells overexpressing ATF3 than in the control cells. In addition, MMP2 and MMP9 expression was found to be down-regulated in HCT116 cells overexpressing ATF3 at both translational and transcriptional levels, and Western blotting detected up-regulation of TIMP-2 and TIMP-1 expression in HCT116 cells overexpressing ATF3 relative to the control cells. Overexpression of ATF3 resulted in reversal of epithelial-mesenchymal transition (EMT), as revealed by changes of cell morphology and altered expression of E-cadherin, Snail, Slug and Twist. Most importantly, ATF3-overexpressed HCT117 cells produced less and smaller spheres as compared with the control cells. The expression of multiple markers associated with colorectal cancer-initiation including CD24, CD133, CD166, EpCAM, EZH2 and ID1 were down-regulated in HCT116 cells overexpressing ATF3 at both translational and transcriptional levels, while lower beta-catenin protein expression was detected in ATF3-overexpressed HCT116 cells relative to the control cells. The results of this study demonstrate that ATF3 overexpression inhibits colorectal cancer progression and suggest that ATF3 exhibits antitumor activity through reversing EMT and impairing cancer-initiation ability.

OBJECTIVE:To study the effects of caspase recruitment domain membrane-associated guanylate kinase protein 1 (CARMA1) knock-down using RNAi technology on cell proliferation, colony forming, invasion and metastasis of the K562 cells.METHODS:K562 cells with stably-silenced CARMA1 gene was constructed by lentivirus-mediated RNAi technology. CARMA1 mRNA and protein levels were respectively detected by reverse transcription PCR (RT-PCR) and Western blotting. Cell proliferation was analyzed by trypan blue exclusion; colony forming was detected by colony forming assay; invasion and metastasis in vitro were determined with Transwell® assay with or without matrigel. Signaling pathway molecules were detected by RT-PCR and Western blotting.RESULTS:Six cell lines with knocked-down gene were successfully constructed. Of them, one was negative control (K562/sh-eGFP), and the other five were CARMA1-silenced cells. K562/shCARMA 1-93 showed the greatest inhibition of CARMA 1 gene and protein expressions. Trypan blue exclusion and colony forming assay showed that the ability of cell proliferation and colony forming were significantly suppressed in K562/shCARMA 1-93 (P<0.01). Compared with blank control K562 and negative control K562/sh-eGFP, the growth inhibition rates were respectively 29.3% and 28.6%; the colony forming inhibition rates were respectively 37.6% and 34.1%; Transwell® assay with or without matrigel showed that the number of K562/shCARMA1-93 which passed through the membrane was obviously lower than that of the control group (P<0.01). The expressions of nucleic transcription factor-κB (NF-κB) and early growth response 1 (EGR1) were reduced following the inhibition of CARMA1.CONCLUSION:CARMA1 gene knock-down had an impact on cell proliferation, colony forming, invasion and metastasis of K562 cells, which is possibly related to the inhibition of NF-κB and JNK/EGR-1 signaling pathways.

Upregulation of nuclear factor-κB (NF-κB) in colorectal carcinoma (CRC) accelerates tumor growth, whereas, irinotecan (CPT-11)-induced NF-κB activation reduces chemosensitivity and weakens the anti-colorectal cancer function itself, while proteasome inhibitors can inhibit NF-κB and improve the effect of chemotherapy. Carfilzomib (CFZ) is a novel proteasome inhibitor that has been recently approved by the FDA and is in clinical use for the treatment of multiple myeloma, but little is known about its activity against CRC. The aim of the present study was to explore whether CFZ alone or in combination with CPT-11 is effective in CRC treatment. We evaluated the novel therapeutic ability and mechanism of action of CFZ in CRC in vitro and in vivo. SW620 cells were incubated with CFZ alone or in combination with CPT-11. Cell proliferation was assessed by WST-1 and clonogenic assays, the cytotoxic interaction was assessed with a combination index (CI). Cell cycle progression was analysed with flow cytometry. Cell apoptosis was evaluated by detecting the Annexin V/propidium iodide (PI) ratio, caspase 3 and CD95 expression, and with TUNEL staining. Cell migration and invasion was determined with a wound-healing assay and a Transwell matrix penetration assay. A CRC xenograft model was established to monitor tumor growth. EMSA was used to analyse NF-κB activation and western blot analysis was used to detect the protein levels of related signaling factors. CFZ significantly inhibited the growth of SW620 cells, and had synergistic inhibitory effects with CPT-11 on survival and colony formation; possibly by inhibition of NF-κB activation, MEK/ERK and PI3K/AKT pathway factor dephosphorylation and survivin downregulation. Co-administration of CFZ and CPT-11 induced G2/M arrest, increased p21WAF1/CIP, and decreased mutant p53 and cdc25c expression. Induction of apoptosis was accompanied by marked increases in PARP cleavage, caspase 3 activation, an increase of CD95 and p-p38, and ATF3 activation. Combination treatment lowered the invasive and migration ability of SW620 cells, reduced MMP and increased TIMP protein expression. Finally, co-administration of CFZ and CPT-11 suppressed tumor growth and increased apoptosis compared with single-agent treatment in SW620 xenograft models correlated with NF-κB downregulation. Carfilzomib alone or in combination with CPT-11 is effective against colorectal cancer through inhibition of multiple mechanisms related to NF-κB, and could be a potential novel therapy for CRC.